INTRODUCTION: Mast cells, when stimulated by antigen via the high affinity receptor for IgE (Fc{epsilon}RI), release inflammatory factors by three mechanisms, degranulation, rapid synthesis of inflammatory lipids (eicosanoids) derived from arachidonic acid, and the generation of cytokines via gene transcription. Our general objective is to delineate the signaling pathways for each of these mechanisms and determine how other stimulants and therapeutic agents interact with these pathways. We have shown that degranulation is regulated primarily by changes in cytosolic Ca2+ (the calcium signal), protein kinase (PK) C, and phospholipase (PL) D (see accompanying report HL000993-19 LMI) whereas the generation of eicosanoids is dependent on a calcium signal and activation of mitogen activated protein (MAP) kinases for activation of PLA2 and induction of downstream enzymes such as cyclooxygenase-2 (COX-2) and 5-lipoxygenase. The generation of cytokines is driven by induction/activation of a variety of transcription factors that are regulated by Ca2+, PKC, MAP kinases, and other signaling pathways. Some of these signalling processes as well as release of inflammatory mediators are potently suppressed by glucocorticoids while some are enhanced, additively or synergistically, by co-stimulation of mast cells via Fc{epsilon}RI and receptors for adenosine (A3), stem cell factor (Kit), or Toll-like receptors (TLRs)(see previous report in this series). A guiding theme is that the mast cell in situ is activated by antigen in the context of physiologic (e.g., stem cell factor) and pathologic (e.g., adenosine and TLR ligands) factors and that it is also necessary to study Fc{epsilon}RI-mediated signals in that context. CURRENT OBJECTIVES: This year we have focused on the mechanisms for the synergistic interactions of antigen with other mast cell stimulants such as stem cell factor (SCF)and pathogenic ligands of Toll-like receptors (TLRs) and show that in combination the stimulants activate a wider array of cytokine-related transcription factors than the individual stimulants and thus enhance cytokine production. We have continued investigating the mechanisms by which glucocorticoids suppress mast cell activation. In doing so, we identified two key signaling events that are disrupted by dexamethasone, our prototypic glucocorticoid, and a mechanism by which these events are disrupted. Studies were conducted with a variety of mast cell lines including primary cultures of bone marrow-derived mast cells (BMMC)and human mast cells to determine whether or not our findings applied to all subtypes of mast cells. The new findings are as follows. SYNERGISTIC INTERACTIONS BETWEEN ANTIGEN AND SCF. Studies with SCF completed this past year showed that SCF failed to induce degranulation but acted in synergy with antigen to markedly enhance degranulation, release of arachidonic acid, as well as production of cytokines. The explanation for these varied responses was that while antigen activated PKC and PLC-gamma/calcium mobilization, two critical signals for degranulation, SCF failed to activate PKC and degranulation. However, SCF stimulated the PLC-gamma/calcium pathway and together with antigen enhanced this response and degranulation. Both SCF and antigen induced signals that are associated with cytokine production namely, activation of MAP kinases, phosphatidylinositol (PI) 3-kinase, and the transcription factors, NF-kappa-B and NFAT. In addition, SCF stimulated phosphorylation of STAT5 and STAT6 while antigen stimulated the PKC-dependent induction of the AP-1 components, c-Jun and c-Fos. Together, the stimulants engaged a wider and more effective repertoire of transcripton factors to achieve optimal conditions for generation of inflammatory cytokines. Interestingly, most signals were down-regulated on continuous exposure to SCF but were reactivated along with cytokine gene transcription on addition of antigen. This down regulation might limit constitutive production of inflammatory cytokines under normal physiologic conditions where continuous exposure to SCF is essential for survival and maturation of mast cells (Ref. 1). GENERATION OF CYTOKINES ON STIMULATION OF MAST CELLS VIA TLRs: In addition to mediating IgE-dependent allergic reactions via Fc{epsilon}RI, mast cells may play a critical protective role against acute bacterial and parasitic infections by production of inflammatory cytokines through the activation of TLRs. We find that antigen interacts synergistically with TLR2/TLR1, TLR2/TLR6, and TLR4 ligands to markedly enhance (up to 40-fold) production of inflammatory cytokines in primary and tumor murine mast cell lines. However, the TLR ligands neither stimulated degranulation and release of arachidonic acid nor influenced such responses to antigen probably because these ligands failed to generate a necessary calcium signal. The enhanced cytokine production could be attributed to synergistic activation of mitogen activated protein kinases in addition to the engagement of a more effective repertoire of transcription factors for cytokine gene transcription. For example, antigen but not TLR ligands activated NFAT and AP1 components whereas TLR ligands activated STAT 1 but together with antigen enhanced activation of AP1. The synergistic interactions of TLR ligands and antigen might have relevance to the exacerbation of IgE-mediated allergic diseases by infectious agents (Ref. 2). IDENTIFICATION OF PRIMARY SITES OF ACTIONS OF GLUCORTICOIDS: We find that treatment with dexamethasone disrupts the interaction of PI 3-kinase with the adaptor protein, Gab2, and as a consequence downstream events that lead to degranulation and transcription of antigen-inducible cytokines (Ref. 3). Prior studies had shown that dexamethasone also disrupts the interaction of Raf1 with Ras and as consequence activation of the Erk (Cissel and Beaven, J. Biol. Chem. 275:7066, 2000). These actions are of slow onset (12 hrs for maximum effect), of long duration (24 hrs or more), and apparent with clinically relevant concentrations of dexamethasone (1-10 nM). Studies with other steroids (refs 3 and 4) and expressed mutants of the glucorticoid receptor(unpublished)indicate that disruption was mediated through interaction of the glucorticoid receptor with glucocorticoid response elements (GRE), a process referred to as "transactivation". The mechanism for these disruptions was revealed by Gene chip analysis. After treatment with dexamethasone there is a substantial increase in mRNA and protein of several inhibitory regulators. One is the "downstream of tyrosine kinase" (Dok)-1, which is known to inhibit Ras activation by inhibiting the Ras GTPase-activating protein (RasGAP)in B cells (Ref. 4). Another was the "Src-like adaoptor protein", SLAP, which inhibits Syk activity in B cells (unpublished). By co-expression of wild-type and mutant proteins as well as use of siRNA technology we showed that the induction of these proteins suppressed activation of Ras and Syk, their engagement with Raf-1 and Gab2 respectively, and downstream events that lead to degranulation. The upregulation of these inhibitory regulators was of slow onset and apparent with 1 nM to 10 nM dexamethasone--features that are reminiscent of the inhibition of the Ras/Raf-1/Erk and Gab2/PI 3-kinase pathways by dexamethasone. These findings suggest that the current paradigm for the the anti-inflammatory actions of glucocorticoids, the suppression of cytokine gene transcription by "transrepression", must be broadened to include induction of inhibitory regulators by gene "transactivation". This has significant bearing on current interest in the development of "disociated" steroids which lack the "transactivation" potential of current glucococorticoids which was thought to account for the undesirable effects of glucocorticoids.